ES2642088T3 - Compositions and methods of using ABCI chondroitinase mutants - Google Patents

Description

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Chondroitinase can be obtained from several sources, including a microorganism that naturally expresses a chondroitinase; for example, but not limited to, E. coli, Proteus vulgaris, or from the expression of a recombinant protein in a host cell. The host cell can be a prokaryotic cell (such as E. coli) or a eukaryotic cell (such as a yeast, a mammalian cell or an insect cell).

The ABCI mutant chondroitinase nucleic acids of the present invention can be obtained by various methods known in the art. For example, the polymerase chain reaction and / or other techniques can be used to generate mutations in wild-type P. vulgaris or another chondroitinase coding sequence. A method of obtaining a nucleic acid sequence encoding an ABCI chondroitinase mutant enzyme selected from 055D2-3 (SEQ ID NO: 1), 079B6-2 (SEQ ID NO: 2), 023G6-4 (SEQ ID NO: 5 ) and 005B12-3 (SEQ ID NO: 6). The invention may include a method of obtaining a nucleic acid sequence of the invention, wherein the nucleic acid is selected from nucleic acid 055D2-3 (SEQ ID NO: 9), nucleic acid 079B6-2 (SEQ ID NO: 10 ), nucleic acid 023G6-4 (SEQ ID NO: 13) and nucleic acid 005B12-3 (SEQ ID NO: 14).

Expression of a recombinant mutant ABCI nucleic acid sequence of the invention can be accomplished by ligating a nucleic acid encoding the ABCI mutant protein, or a portion thereof, in a vector suitable for expression in prokaryotic cells, or in eukaryotic cells. , or both. The procedures for ligation are well known to professionals with normal experience in the art. Expression vectors for the production of recombinant forms of the subject chondroitinase polypeptides include plasmids and other vectors. For example, suitable vectors for the expression of an ABCI mutant chondroitinase polypeptide include plasmids of the types: plasmids derived from pBR322, plasmids derived from pEMBL, plasmids derived from pEX, plasmids derived from pBTac and plasmids derived from pUC for expression in cells prokaryotes, such as E. coli.

Several vectors exist for the expression of recombinant proteins in yeast and could be used to express a recombinant ABCI mutant protein of the invention. For example, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are useful cloning and expression vehicles in the introduction of genetic constructs in S. cerevisiae (see, e.g., Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. lnouyc Academic Press, p. 83, incorporated by reference in this document.

An ABCI chondroitinase mutant polypeptide of the invention can be produced recombinantly using an expression vector generated by subcloning the coding sequence of one of the chondroitinase proteins represented in 055D2-3 (SEQ ID NO: 1), 079B6-2 ( SEQ ID NO: 2), 023G6-4 (SEQ ID NO: 5) and 005B12-3 (SEQ ID NO: 6).

In some cases, it may be desirable to express a recombinant ABCI chondroitinase mutant polypeptide of the invention by using an insect expression system such as the baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393 and pVL941), vectors derived from pAcUW (such as pAcUW1), and vectors derived from pBlueBac (such as pBlueBac III containing β -gal ).

Expression vectors and host cells listed herein are provided by way of example only and represent the well known systems available to those skilled in the art, which may be useful for expressing nucleic acid molecules. The professional with a normal experience in the art will be aware of other systems suitable for the propagation of maintenance or the expression of the nucleic acid molecules described herein.

The enzymes of the invention can be formulated in pharmaceutical compositions and formulations. Suitable stable formulations and purification methods are set forth in PCT application No. US2005 / 017464 pending in common with this, filed on May 18, 2005 and entitled "Methods of Purifying Chondroitinase and Stable Formulations Thereof" ("Methods of Chondroitinase purification and stable formulations thereof ”).

Several embodiments provide a stable formulation of an ABCI mutant chondroitinase enzyme of the invention, both for storage and for administration. Generally, the enzyme of such stable formulations shows at least about 50% activity at about 24 hours, preferably at least about 75% activity, more preferably at least about 85% activity. In another aspect of the invention, the formulations consistently provide a stable chondroitinase activity.

In one embodiment, the chondroitinase is formulated in a phosphate buffer, preferably a sodium phosphate buffer with a concentration in the range of about 50 mM to about 1 M. A preferred embodiment is about 750 mM sodium phosphate. Another preferred embodiment is approximately 100 mM sodium phosphate. In another embodiment, chondroitinase can be formulated in a sodium phosphate buffer further comprising sodium acetate. Sodium acetate may be present in the range of 25 mM to approximately 75 mM. In a preferred embodiment, the concentration of sodium acetate is approximately 50 mM. In one embodiment, a preferred formulation for administration is a chondroitinase in a buffer with

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For example, in some aspects, the invention is directed to a pharmaceutical composition comprising a compound, as defined above, and a pharmaceutically acceptable carrier or diluent, or an effective amount of a pharmaceutical composition comprising a compound as defined above.

The compounds of the present invention can be administered in the conventional manner by any route in which they are active. Administration can be systemic, topical, or oral. For example, administration may be, but not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, oral, or ocular, or intravaginally, by inhalation, by injection of deposit, or by implants. Thus, the modes of administration for the compounds of the present invention (either alone or in combination with other pharmaceutical products) may be, but not limited to, sublingual, injectable (including short-acting, depot, implant and pill forms. injected subcutaneously or intramuscularly), or through the use of vaginal creams, suppositories, vaginal ovules, vaginal rings, rectal suppositories, intrauterine devices and transdermal forms, such as patches and creams.

The specific modes of administration will depend on the indication. The selection of the specific route of administration and the dose regimen has to be adjusted or assessed by the physician according to methods known to the clinician in order to obtain the optimal clinical response. The amount of compound to be administered is that amount that is therapeutically effective. The dose to be administered will depend on the characteristics of the subject being treated, e.g. eg, the particular treated animal, age, weight, health, types of concurrent treatment, if any, and the frequency of treatments, and can easily be determined by a professional skilled in the art (for example , by the doctor).

Pharmaceutical formulations containing the compounds of the present invention and a suitable carrier may be solid dosage forms that include, but are not limited to, tablets, capsules, seals, pills, pills, powders and granules; topical dosage forms, including, but not limited to, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, ointments, pastes, creams, gels and jellies, and foams; and parenteral dosage forms that include, but are not limited to, solutions, suspensions, emulsions, and dry powder; which comprises an effective amount of a polymer or copolymer of the present invention. It is also known in the art that active ingredients may be contained in such formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surface active agents, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like The means and methods for administration are known in the art and an expert can consult various pharmacological references for guidance. For example, see Modern Pharmaceutics, Banker and Rhodes, 4th Ed., Informa Healthcare (2002); and Goodman and Gilman´s The Pharmaceutical Basis of Therapeutics, 10th ed., McGraw-Hill (2001).

The compounds of the present invention can be formulated for parenteral administration by injection, e.g. e.g., by bolus injection or continuous infusion. The compounds can be administered by continuous infusion subcutaneously over a period of about 15 minutes to about 24 hours. Formulations for injection may be presented as a unit dose, for example in ampoules or in multiple dose containers, with a built-in preservative. The compositions may take forms such as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents.

For oral administration, the compounds can be easily formulated by combining said compounds with pharmaceutically acceptable carriers well known in the art. Such vehicles allow the compounds of the invention to be formulated in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, pastes, suspensions and the like, for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the granule mixture, after adding suitable auxiliary agents, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol and sorbitol; cellulose preparations such as, but not limited to, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablets or coatings of the dragees for identification or to characterize different dose combinations of active compound.

Pharmaceutical preparations that can be used orally include, but are not limited to, hard capsules made of gelatin, as well as soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Pressure adjustment capsules may contain the active ingredients mixed with such a load.

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then with an equal volume of chondroitin C sulfate of 0.25 mg / ml (Sigma), a cABCI substrate that results in the cleavage of GAG chains. After incubation at room temperature of 10 minutes, DMB reagent was added, and the absorbance at 660 nm was measured. Positive identifications with absorbance measures greater than the wild-type enzyme on the same plate were counted as positive successes, indicating

5 increased activity after thermal stress.

Creation of the recombined library: The ten most thermally resistant clones of modules A, B and C were randomly recombined to produce a combinatorial product library. The PCR products of each module were combined in an equimolar relationship, with a molar equivalent of the corresponding wild type also present. This created a group of 9 variant sequences for Module C, and a cluster of 10 11 variants for both modules A and B. A 3-way ligation was performed in which each module could only be linked in the correct orientation with the appropriate flanking module or modules and ligated into the pET15b vector DNA to produce expression clones containing full length cABCI. The total size of this library is 1089 variants cABCI sequences. The number of colonies obtained containing the correct clonic structure was at least 5 times the expected number of individual mutant genes. The ligation was weighted to

15 produce mostly clones containing two or three mutant modules, thus creating new combinations of the mutations identified in the "hits" (identifications) of the initial screening.

EXAMPLE 2

The enzymes referred to by SEQ ID Nos. 1, 2, 5 and 6 of the present example illustrate by way of example chondroitinase mutant enzymes of the present invention.

20 It was confirmed that thermally stable mutants are generated through the molecular evolution process. The modified DMB assay identified the clones with the highest thermal stability at 42 ° C for 2 hours, compared to wild-type cABCI. It is likely that stability at this temperature confers greater stability at 37 ° C, allowing ease of handling and supply for in vivo studies, since permanent mini-pumps could be used for dosing. The individual modules had as

25 results in an expected margin of positive identifications globally, as defined by the study parameters.

Clones that have greater thermal stability were characterized by sequencing. All nucleotide and amino acid sequences are indicated as the wild type and then the mutant version (wild type to mutant).

Chondroitinase mutant enzyme

Sequence of ABCI mutant chondroitinase Sequence of

ABCI (protein)

amino acids (nucleic acid) nucleotides

055D2-3 protein

E256 to K256 055D2-3 nucleic acid T450 to C450

(SEQ ID No. 1)

(SEQ ID NO. 9) G766 to A766

C2295 to T2295

079B6-2 protein (SEQ ID No. 2)

D683 to N683 Nucleic acid 079B6-2 (SEQ ID No. 10) G2047 to A2047

Protein 079D2-2 (SEQ ID No. 3)

Nucleic acid 079D2-2 (SEQ ID NO: 11) A1773 to G1773 G1980 to A1980 T2068 to C2068 A2076 to G2076

057G1-1 protein (SEQ ID No. 4)

Nucleic acid 057G1-1 (SEQ ID No. 12) G483 to A483 T1110 to C1110 T1821 to C1821

023G6-4 protein (SEQ ID No. 5)

I919 to F919 A736 to P736 Nucleic acid 023G6-4 (SEQ ID No. 13) A2755 to T2755 G2206 to C2206

Protein 005B12-3 (SEQ ID No. 6)

E296 to K296 Nucleic acid 005B12-3 (SEQ ID No. 14) G985 to A985

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to A232. The samples were subjected to a thermal stress of 37 ° C in a humidified incubator. Activity readings were taken every day until the native sample lost all activity. The test of the remaining samples continued 3 times per week. The activity readings shown as a percentage of the total activity retained for a few variants are presented in Figure 3 and in Tables 2A and 2B below.

Table 2A: Time (days) at 37 ° C

0.50

1.50 2.50 3.79 4.79 5.79

cABCI

56.43 34.45 25.48 14.42 8.63 5.77

023G6-1

58.32 42.28 41.70 31.96 24.61 19.82

0.79B6-2

65.23 54.21 44.96 35.14 19.09 17.93

purified cABCI

33.78 18.64 11.21 4.51 1.59 0.66

Table 2B:

6.79

8.79 11.79 13.79 15.79 18.79

cABCI

3.55 2.23 0.74 0.00 0.00 0.00

023G6-1

15.23 12.41 9.00 6.74 3.76 1.86

0.79B6-2

13.89 10.76 5.02 2.89 1.51 1.10

purified cABCI

0.00 0.00 0.00 0.00 0.00 0.00

EXAMPLE 5

10 Stability of mutant chondroitinase after UV treatment. After growth and expression, a chondroitinase mutant is extracted using BPER (Pierce) as above and exposed to UV light. Chondroitin lyase activity is measured by a DMB assay.

Claims (1)

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